1. Field of the Inventive Concept(s)
The presently disclosed and claimed inventive concept(s) relates generally to biocidal formulations that utilize free radical generation as a mechanism of toxicity, and more specifically, to selenium-based formulations that utilize free radical generation as a mechanism of toxicity.
2. Description of the Background Art
Selenium (Se) is among the most toxic of all known minerals. Throughout the period of time between the discovery of selenium toxicity in livestock in 1934 and 1988, many hypotheses were put forth to explain the mechanism by which many but not all compounds of selenium were toxic. None of these theories of selenium toxicity proved satisfactory in fully explaining why selenium was toxic. In 1989, Seko et al. (In: Proceedings of the fourth international symposium on selenium and medicine (ed., Wendel, A.) pp. 70-73, Springer-Verlag, Heidelberg, Germany, (1989)), reported that selenite, (SeO3), an inorganic form of Se, reacted with a thiol, glutathione, (GSH), to produce superoxide (O2−). Since superoxide is a known toxicant, this raised the possibility that all selenium compounds that are toxic might generate superoxide. Through the testing of many selenium compounds, it was found that the inorganic compounds, SeO3 and selenium dioxide (SeO2) were able to generate O2− and hydrogen peroxide (H2O2) when presented with a thiol, such as glutathione, cysteine (CysSH), or dithiothreitol D(SH)2. Furthermore, it was found that all diselenides tested of the composition RSeSeR likewise would generate O2− and H2O2 when presented with any of the before mentioned thiols.
In 1947, Feigl et al. (Analytical Chemistry, 19:351-353 (1947)), reported that selenium could catalyze a redox reaction involving sulfide oxidation. This soon became a common test for selenium using methylene blue. This reaction was further studied by others using different selenium compounds and thiols, demonstrating catalysis for some but not all selenium compounds. See, West et al. (Analytic Chemistry, 40:966-968 (1968)); Levander et al. (Biochemistry, 12:4591-4595 (1973)), Rhead et al. (Bioorganic Chemistry, 3:225-242 (1974)). The selenium catalytic activity of selenocystine (RSeSeR) in the presence of thiols was reported in 1958. It is now believed that all of the foregoing reactions of selenium compounds produce superoxide. See, Xu et al. (Advances in Free Radical Biology and Medicine, 1:35-48 (1991)); Xu et al. (Huzahong Longong Daxus Xuebao, 19:13-19 (1991)); Kitahara et al. (Archives of toxicology, 67:497-501 (1993)); Chaudiere et al. (Archives of Biochemistry and Biophysics, 296:328-336 (1992)).
Selenium and a number of its compounds have been known since the early 1970's to possess anti-cancer properties. It has been generally recognized that selenite and selenium dioxide are good anti-cancer agents in vitro and in experimental animals and that the compounds are also cytotoxic to both cancer and normal cells in vitro. U.S. Pat. No. 5,104,852 issued to Kralick et al. describes the use of selenodiglutathione (GSSeSG) and other selenodithiols of the configuration (RSSeSR) to treat cancer. Selenodiglutathione is the product of the reaction between selenite or selenium dioxide with glutathione. The compound, selenodiglutathione, has been isolated. U.S. Pat. No. 5,104,852, however, does not describe the mechanism of action by which selenodiglutathione and like compounds are useful in treating cancer.
In 1982, the interaction of selenite and selenocystine with glutathione in the cytotoxicity and lysis of rat erythrocyte membranes was described by Hu et al. (Biochemical Pharmacology, 32:857-961 (1983)). This cytotoxicity, as revealed by scanning electron microscopy of rat erythrocytes, caused the erythrocyte membranes to become burred, the cells to quadruple in size and lyse similar to that described by Kellogg et al. (J. Biol. Chem., 252:6721-6728 (1977)). This toxicity, however, was not expressed by selenomethionine, a compound possessing the configuration RSeCH3. In 1991, an article by Yan et al. (FASEB J., 5:A581 (1991)), showed a dose responsive toxicity of several selenium compounds to a human mammary tumor cell line. Additional investigations using lucigenin chemiluminescence and luminol chemiluminescence revealed a dose response in O2− and H2O2 generated chemiluminescence by selenite, selenium dioxide and all selenium compounds tested of the configuration RSeSeR. Furthermore, it was found that selenium compounds in the presence of either tumor cells or glutathione alone produced superoxide and H2O2. Chemiluminescence from the reactions of lucigenin with O2− or luminol with H2O2 could be quenched by the native enzymes superoxide dismutase (SOD), catalase (CT) or glutathione peroxidase (GSHPx). Denatured enzymes would not quench these reactions, confirming the generation of the free radical (O2−) and H2O2 by selenium compounds and thiols. All of this selenium free radical chemistry has been reviewed by Spallholz (Free Radical Biology and Medicine, 17:45-64, (1994)).
A summation of this large body of experimental data on selenium toxicity, catalysis and carcinostatic activity is as follows:                1) The selenium compounds, SeO2 and SeO3, react with thiols to produce a selenodithiol of the configuration (RSSeSR). This compound is not toxic per se nor is it carcinostatic. The toxic carcinostatic form of RSSeSR is the reduced selenide anion, RSSe−. This selenopersulfide form of Se is catalytic as shown by the inhibition of both catalysis and superoxide generation by iodoacetic acid and mercaptosuccinic acid.        2) Selenium compounds of the configuration (RSeSeR) or (RSeSeR′) react with thiols to produce the reduced selenite anion RSe− or R′Se−. This selenide ionic form of Se is catalytic as shown by the inhibition of both catalysis and superoxide generation by iodoacetic acid and mercaptosuccinic acid.        3) Organic selenium catalysts of the configuration RSSe−, the selenopersulfide anion, is catalytic in the presence of thiols, and RSSe− continues to generate superoxide (O2−) ion as long as sufficient concentrations of O2− and thiol are in the medium. All of these selenium compounds derived from selenite or selenium dioxide reacting with glutathione (GSH) are converted to elemental selenium (Se.) as follows; SeO3 (SeO2)+2GSH→2GSSeSG→2GSSG+Se.. Elemental selenium (Se.) is non-catalytic and not toxic.        4) Compounds of selenium of the configuration RSe− or RSSe− are toxic due to the catalytic acceleration of thiol oxidation which produces O2−, H2O2 and the more toxic free radical, the hydroxyl radical (.OH). This chemistry had been discussed by Misra (J. Biol. Chem., 249:2151-2155 (1974)) for the spontaneous oxidation of thiols. The association of rapid thiol catalysis by selenium compounds of the configuration RSe− or RSSe− and the toxicity from which it produced free radicals and reactive toxic oxygen products was recognized in 1992 by one of the inventors.        
The use of selenium for the treatment of experimental cancer in animals and cancer in humans in vivo has been extensively described by many authors, such as Milner et al. (“Selenium and transplantable tumors,” (Spallholz, J. E., Martin, J. L., Ganther, H. E., eds.) Selenium in Biology and Medicine, AVI Publishing Co. (1981)); Ip et al. (“Relationship between the chemical form of selenium and anticarcinogenic activity,” CRC Press, Inc., pp. 479-488 (1992)); Caffrey et al. (Cancer Research, 52:4812-4816 (1992)); Schrauzer (Biol. Trace Elem. Res., 33:51-62 (1992)); and Yan et al. (Biochemical Pharmacology, 45:429-437 (1993)). The use of selenium as a cytotoxic agent to both normal cells and cancer cells in vitro for the injection of selenodiglutathione into a tumor mass to kill tumor cells has been described in U.S. Pat. No. 5,104,852, issued to Kralick et al. In U.S. Pat. No. 4,671,958, Rodwell et al. described many antibacterial drugs, 3 antiviral drugs, 1 antifungal drug, 7 antineoplastic drugs, 3 radiopharmaceuticals, 3 heavy metals and 2 antimycoplasmals as drugs for antibody mediated delivery. The pharmacology for all of these drugs which are listed in Table 1 of U.S. Pat. No. 4,671,958 is generally understood. Table 1 of the Rodwell et al. patent does not contain selenium because its pharmacological action as a free radical generator of (O2−) and other reactive oxygen molecules was not understood or known at that time.
Humans and other animals are in a constant immune-system battle with agents of infectious disease, such as bacteria, viruses, pathogenic fungi and protozoa. A particular problem for healthcare professionals dealing with these infectious agents has been the development of antibiotic resistant bacteria, which are refractory to many of the antibiotic agents that initially promised to provide a reliable cure.
As a result of widespread public concern with infectious bacteria, antimicrobial treatment of materials such as fabrics, fibers, polymers and even children's toys have become increasingly popular. While the demand for such antimicrobial articles is high, relatively few types of such articles are available, and not all of those available are both effective against a broad spectrum of bacteria and capable of sustained antimicrobial activity without being released into the environment or being gradually chemically inactivated.
Research and development of durable functional fibers has advanced in recent years, including new methods of incorporating antibiotics as bactericidal agents directly into the fibers. The chemical and medical literature describes many compounds that have antimicrobial activity. Although the mechanism of action of these antimicrobials varies, they generally function by one or more of the following manners: inhibition of cell wall synthesis or repair; alteration of cell wall permeability; inhibition of protein synthesis; and/or inhibition of the synthesis of nucleic acids (DNA or RNA).
At least since the 1870s, silver ions have been recognized as an antibacterial agent, and have been particularly noted for their ability to resist the development of drug-resistance in target bacteria. In general, silver cations (Ag+) are thought to possess antimicrobial activity because they are highly reactive chemical structures that bind strongly to electron donor groups containing sulfur, oxygen, or nitrogen that are present in microbial targets. The biological target molecules generally contain all these components in the form of thio, amino, imidazole, carboxylate, and phosphate groups. Silver ions act by displacing other essential metal ions such as calcium or zinc. The direct binding of silver ions to bacterial DNA may also serve to inhibit a number of important transport processes, such as phosphate and succinate uptake, and can interact with cellular oxidation processes as well as the respiratory chain. The silver ion-induced antibacterial killing rate is directly proportional to silver ion concentrations, typically acting at multiple targets. Indeed, for silver ion-based antimicrobial articles and devices to be effective as antimicrobial vectors, the silver ions with which they are impregnated must be slowly released into the environment so that they are free to contact and inhibit the growth of destructive microbes in the environment. Accordingly, the antimicrobial activity of silver-coated and silver-impregnated articles and devices is dependent upon the controlled release rate of the unbound, free silver ions they carry, and the continued antimicrobial efficacy of such silver-based antimicrobials is necessarily limited by the supply of free silver ions they retain.
The inventor's previous work, as disclosed and claimed in U.S. Pat. Nos. 5,783,454; 5,994,151; 6,033,917; 6,040,197; 6,043,098; 6,043,099; and 6,077,714; all issued to Spallholz et al., discloses methods for making selenium-carrier conjugates by covalently attaching (i) an organic selenium compound selected from the group consisting of RSeH, RSeR′, RSeSeR and RSeSeR′, wherein R and R′ each comprise an aliphatic residue containing at least one reactive group selected from the group consisting of aldehyde (ketone), amino, alcoholic, phosphate, sulfate, halogen, alkane, alkene, alkyne or phenolic reactive groups and combinations thereof, to (ii) a carrier having a constituent capable of forming a covalent bond with said reactive groups of said selenium compound to produce a selenium-carrier conjugate which is capable of specific attachment to a target site. The carrier may be a protein, such as an antibody specific to a bacteria, virus, protozoa, or cell antigen, including without limitation, cell surface antigens, a peptide, carbohydrate, lipid, vitamin, drug, lectin, plasmid, liposome, nucleic acid or a non-metallic implantable device, such as an intraocular implant or a vascular shunt.
The '454 patent demonstrates the cytotoxicity of selenofolate of the configuration Folate-SeR, which produces superoxide in the presence of glutathione or other thiols, as measured by lucigenin chemiluminescence; this modified vitamin compound is cytotoxic to cancer cells upon uptake in a dose dependent manner. The '454 patent also demonstrates the ability of selenocystamine attached to plastic or a cellulose matrix to inhibit cellular growth.
The selenium-carrier conjugates of the prior art (as taught in the various patents listed above) require covalent attachment of the selenium compound to the carrier molecule in order to be effective. In addition, the leaving groups generated when RSe— is produced, as taught by the prior art, are toxic. Therefore there is a need for sustainable and effective biocidal agents that both avoid the formation of resistant microbes and can be adapted for use in manufacturing materials, and in application to solid substrates, which overcome the disadvantages and defects of the prior art. It is to such improved biocidal compositions, and methods of production and use thereof, that the presently disclosed and claimed inventive concept(s) is directed.